Analysis of FANCA-deficient and FANCA-expressing IPSC-derived hematopoietic cells. (A) Total RNA was isolated from the nonadherent cells at day 8 EHT, and expression of FANCA was analyzed by quantitative PCR (n = 3 biologic replicates; results presented as mean ± SEM, analyzed by a Student t test). (B-C) Nonadherent cells were isolated at day 8 of EHT after culture with or without doxycycline and then cultured with mitomycin C (20 ng/mL) for 18 hours, at which time the cells were fixed and immunostained for FANCD2 and γH2AX and visualized by confocal microscopy (scale bar, 10 μm). Results are compiled from 3 separate lines, and the proportion of cells with double-positive foci presented as mean ± SEM, analyzed by a paired Student t test. (D-E) A total of 10 000 nonadherent cells collected from day 5 or day 8 EHT cultures were embedded into methylcellulose with hematopoietic growth factors. Doxycycline was either maintained in these assays or withheld consistent with EHT culture conditions. After 14 days, colonies were scored morphologically, and the results were quantified (n = 7 biologic replicates for day 5 and n = 10 biologic replicates day 8 across 3 cell lines; total colony numbers analyzed by a paired Student t test, results are presented as mean proportions of each colony type ± standard deviation). (F) Hematopoietic colonies were isolated at day 14 from cultures with doxycycline and spun onto slides when the cells were stained with May-Grünwald-Giemsa and analyzed by light microscopy (scale bar, 10 μm).